Expression of TGF-beta-like molecules in the life cycle of Schistosoma japonicum.

The transforming growth factor beta (TGF-beta) family controls an extremely wide range of biological activities, such as the growth and differentiation of cells, and immunological events against infectious agents. Although TGF-beta homologs appear to be widely present in metazoan animals, studies of parasite-derived molecules are relatively few. Using antibodies against anti-mouse TGF-beta1, -beta2, and -beta3, we show the expression of TGF-beta-like molecules in Schistosoma japonicum cercariae, schistosomula, eggs and adult worms. Intense immunoreactivity was found on the surface of free-living cercarial bodies. In transverse sections of cercariae, the molecules were localized in the tegument and subtegumental cells, and the number and distribution of producing cells significantly differed with each antibody. In the skin-migrating stage, the expression in the tegumental surface gradually decreased and became almost negative within 48 h of exposure. In adult worms and eggs, the reactivity was found in subtegumental cells and in cells of a tubular structure, respectively. In western blot analysis, the detection of conventional TGF-beta molecules failed. The expression of TGF-beta-like molecules was distinctly regulated at each developmental stage.

Basal membrane localization of MRP1 in human placental trophoblast.

The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta. ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics. In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp. Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively. Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side. These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast. MRP1 was also detected on the abluminal side of blood vessels in the villi. Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.

The spatiotemporal relationship among Schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats.

To morphologically define the aging-related features during muscle reinnervation the spatiotemporal relationships among the major components of the neuromuscular junctions (NMJs) were investigated. A total of 64 rats, 30 adults (4 months old) and 34 aged adults (24 months old), were used. Between 1 and 12 weeks after sciatic nerve-crushing injury, cryosections of skeletal muscle were single or double labeled for S100, a marker of Schwann cells (SCs), for protein gene product 9.5, a neuronal marker, and for alpha-bungarotoxin (alpha-BT), a marker of the acetylcholine receptor site (AChR site), and then observed by confocal laser microscopy. The most obvious age changes were noted: (1) the regenerating SCs and axons were delayed in their arrival at the NMJ, (2) the dimensions of terminal SCs and AChR sites displayed a drastic and long-lasting drop (for terminal SCs, during 1-8 weeks; for AChR sites, during 1-12 weeks); (3) the degree of spatial overlap between AChR sites and terminal SCs was markedly low until 8 weeks post-crush; (4) damage and poor formation in the SCs, terminal axons and AChR sites, together with poor process extension from the terminal SC or terminal axon, were pronounced; (5) persistent aberrant changes, such as multiple innervation and terminal axon sprouting, together with poorly formed collateral innervation, nerve bundles, and NMJs, more frequently occurred in the later reinnervation period. Thus, with aging, regeneration is impaired during the period in which regenerating SC strands and axons extend into NMJs and the subsequent establishment of nerve-muscle contact is in progress. A complex set of morphological abnormalities between or among the TSCs, terminal axons, and AChR sites may be important in slowing of regeneration and reinnervation in aged motor endplates.

A novel protein interacts with a clock-related protein, rPer1.

Mammalian Per proteins are thought to be important in the mechanism of circadian rhythm. We identified a novel protein PIPS (Per1 interacting protein of the suprachiasmatic nucleus) with the yeast two-hybrid system using PAS domain of rat Per1 (rPer1) as a bait. PIPS is about a 180-kDa protein and expressed mainly in the brain, especially in the hypothalamus including the suprachiasmatic nuclei (SCN). PIPS interacts with mouse Per1 (mPer1) in vitro and in cultured cells transfected with both molecules. Furthermore, it was found that mPer1 translocated PIPS into the nuclei in the cultured cells. Thus, these findings suggest a possibility that PIPS is involved in the feedback loop or output mechanism of circadian rhythm through interacting with Per1 in the SCN.

Histology of intervertebral disc protrusion: an experimental study using an aged rat model.

STUDY DESIGN: In vitro experimental intervertebral disc ruptures of aged rats were examined histologically. OBJECTIVES: To clarify the mechanism of intervertebral disc herniations by microscopic investigation of ruptured discs. SUMMARY OF BACKGROUND DATA: Clinically, disc herniations have been classified into two types: extrusion and protrusion. However, the pathogenesis of protrusion type herniations has not yet been demonstrated by any studies. To clarify this issue, it is essential to establish an appropriate model producing disc herniations, and to examine the sequential changes in the structure of herniated discs. METHODS: Lumbar discs of 2-year-old rats were examined histologically and compared with human lumbar discs. To examine structural changes in discs subjected to repetitive motion stress, 400 repetitions of a sequence of flexion (30 degrees ) and axial rotation (6 degrees ) were applied in vitro to the lumbar discs of the animals. RESULTS: The microstructure of normal lumbar discs in aged rats was similar in many ways to the human lumbar discs in a 20- to 40-year-old adult. Of 10 discs subjected to repetitive stress, 4 were ruptured at the junction between the posterior anulus fibrosus and the sacral cartilage endplate. One had an extruded nucleus pulposus, and three had a protruded anulus fibrosus, which displayed disorganized structure containing widened and flaccid lamellae. CONCLUSIONS: The results from this study indicate that disc protrusion can be caused by disorganization of the ruptured annular lamellae, not by focal compression of the nucleus pulposus.

The hDLG-associated protein DAP interacts with dynein light chain and neuronal nitric oxide synthase.

BACKGROUND: Postsynaptic density (PSD)-95 interacts with and mediates clustering of the N-methyl-D-aspartate-receptors (NMDA-R). PSD-95 also interacts with the hDLG-associated protein DAP, which is also called Synapse-associated protein 90-associated protein (SAPAP), and Guanylate kinase-associated protein (GKAP). RESULTS: DAP interacted directly with the dynein light chain (DLC) family of proteins. DLC was contained in the NMDA-R-PSD-95-DAP-neuronal nitric oxide synthase (nNOS) complex. Furthermore, DAP interacted with nNOS and recruited it into the Triton X-100-insoluble fraction of transfected cells. CONCLUSION: DAP interacts directly with DLC and nNOS, and links these proteins to the NMDA-R-PSD-95 complex.

Morphological features of nerve terminal degeneration as part of the remodeling process in the motor endplate in adult muscles.

With the exception of signs of retraction and withdrawal, there have been few morphological data concerning degenerated neural profiles in adult motor endplates. Here, investigation into the ultrastructure of the soleus motor endplates of adult rats (4 months old) turned up particular axonal degeneration in approximately 3% of the subjects. These axons occur as synaptic debris in the synaptic matrix of the motor endplate, adjacent to thin processes of the perisynaptic cells occupying the outermost layer of the motor endplate and were devoid of basal lamina. They often possessed dense-cored vesicles (50-80 nm). Axonal debris released from Schwann cell processes occurred during the period of acute sciatic neurectomy, when nerve terminals progressively disrupted within the motor endplate-associated Schwann cells. Finally, immunohistochemical staining for antibodies to label macrophages (ED1 or ED2) has shown that nerve fiber-associated macrophages are located near the motor endplate. The results suggest that during the course of endplate remodeling, a few parts of the terminal branches are disposed of through spontaneous collapse, subsequent release from the Schwann cell investment, and eventual ingestion by macrophages in the perisynaptic space.

Heme oxygenase1 (HSP-32) is induced in myelin-phagocytosing Schwann cells of injured sciatic nerves in the rat.

Schwann cells participate in myelin phagocytosis in the early stage of Wallerian degeneration, prior to the recruitment of macrophages. This is the first report that Schwann cells induce heme oxygenase-1 (HO-1), a 32-kDa heat shock protein, only when they have transformed into myelin-phagocytosing cells from myelinating cells (days 2-3) immediately after crush injury of rat sciatic nerves. Double immunofluorescent labelling for HO-1 and transferrin receptors revealed that HO-1-immunoreactive Schwann cells also expressed transferrin receptors suggesting activation of iron metabolism. The transient induction of HO-1 in Schwann cells may contribute to the adaptive function in an altered environment when the cells have lost contact with axons, and may play a crucial role in the ensuing regeneration.

An improved method for avulsion of lumbar nerve roots as an experimental model of nitric oxide-mediated neuronal degeneration.

A root avulsion lesion on the spinal nerve of adult animals is a useful technique to make a model for axotomy-induced motoneuronal degeneration, which is thought to be mediated by nitric oxide (NO). Here, we show a simplified version of extravertebral avulsion in the young adult rat. The L4 nerve always runs under the transverse process of the L5 vertebra, which is located just rostral to the delineation of the iliac crest. We used the iliac crest as a clue for the identification of the L4 nerve during surgery, including before skin incision. In almost all animals the L4 nerve was successfully avulsed at the exit point from the spinal cord. This experimental result was similar to that shown in the previous literature; the number of either Nissl-stained or ChAT-immunoreactive (-ir) motoneurons (MN) gradually decreased, while NOS immunoreactivity was induced in the MN after avulsion. Furthermore, a combined method of confocal laser scanning microscopy and double fluorescent procedures carried out in this model suggested the existence of cellular interaction between NOS-ir MN and OX42-ir or ED1-ir microglia. It is concluded that this simple and fast method of spinal root avulsion is very useful for making a reproducible model of NO-mediated MN cell death, with which the mechanism of neuronal cell death, including neuron-glia interaction, can be further explored.

Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases.

The Src family of protein tyrosine kinases (Src-PTKs) is important in the regulation of growth and differentiation of eukaryotic cells. The activity of Src-PTKs in cells of different types is negatively controlled by Csk, which specifically phosphorylates a conserved regulatory tyrosine residue at the carboxy-terminal tail of the Src-PTKs. Csk is mainly cytoplasmic and Src-PTKs are predominantly membrane-associated. This raises a question about the mechanism of interaction between these enzymes. Here we present Cbp--a transmembrane phosphoprotein that is ubiquitously expressed and binds specifically to the SH2 domain of Csk. Cbp is involved in the membrane localization of Csk and in the Csk-mediated inhibition of c-Src. In the plasma membrane Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane domain, which is important in receptor-mediated signalling. These findings reveal Cbp as a new component of the regulatory mechanism controlling the activity of membrane-associated Src-PTKs.

Identification of tudor repeat associator with PCTAIRE 2 (Trap). A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain.

PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons.

Efferent connections of the anterior hypothalamic nucleus: a biocytin study in the cat.

The efferent connections of the anterior hypothalamic nucleus (AH) were examined using biocytin as anterograde tracer in the cat. The results provide several new findings in addition to confirming earlier observations. In the hypothalamus, the AH projections terminated mainly in the medial regions which are related to the defensive, reproductive and feeding behaviors, and autonomic functions. Moreover, we found dense patches of the AH terminals in the medial preoptic area and ventromedial hypothalamic nucleus, which suggests the existence of modular connections between sub-regions of each nucleus. In addition, the AH projected to regions which may be related to the emotional and autonomic responses, i.e., such regions in the amygdala, midline thalamus, septum, subthalamus, and midbrain. The data suggest that the AH may play an important role in the autonomic functions and behaviors between animals, and thus may play a key role in the defensive behavior elicited in the medial preoptic area and ventromedial hypothalamic nucleus.

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat accessory olfactory bulb.

The nitric oxide cycle consists of nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase to form nitric oxide. We have examined the colocalization of nitric oxide synthase and the cytosolic urea cycle enzymes (argininosuccinate synthetase, argininosuccinate lyase and arginase) in the accessory olfactory bulb of the rat by using a double labeling procedure combining reduced-nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) reaction with fluorescent immunocytochemistry. Each glomerulus showed a different NADPH-d activity, and those with the strongest NADPH-d activities were assembled in the caudomedial part of the accessory olfactory bulb. Argininosuccinate synthetase-like immunoreactive glomeruli were distributed in the caudomedial part of the accessory olfactory bulb, and most of them were also strongly NADPH-d positive. The mitral or tufted cells were argininosuccinate synthetase-, argininosuccinate lyase- and arginase-like immunoreactive, but were not NADPH-d positive. The granule cells were NADPH-d positive or argininosuccinate lyase-like immunoreactive, but were not argininosuccinate synthetase- or arginase-like immunoreactive. Some granule cells were both NADPH-d positive and argininosuccinate lyase-like immunoreactive. The results indicate the heterogeneity of glomeruli of the accessory olfactory bulb with respect to the distribution of these enzymes. The granule cells have nitric oxide synthase and argininosuccinate lyase, and thus may efficiently produce nitric oxide.

Changes in the distribution of peanut agglutinin (PNA) binding molecules during muscle reinnervation following nerve crush injury.

Peanut agglutinin (PNA) staining during muscle reinnervation following a crushing injury of the sciatic nerve was performed in reference to the neural profiles immunolabeled with the PGP 9.5 antibody. PNA staining in the normal controls exhibited dots, granules, or lines along the length of the nerve fibers in the nerve trunk, but was faint or absent in the motor endplate. At seven days post-crush, PNA staining was detected around the vacuolated neural structures in the disorganized nerve trunk, but was still faint or absent in the motor endplate. At twenty-one days post-crush, when PGP 9.5-positive regenerating axons appeared in most of the motor endplates, PNA staining, either faint or strong, followed the pathway of the nerve fibers delineated by PGP 9.5-like immunoreactivity. During reinnervation to the motor endplates, PNA staining displayed signs of remodeling in the nerve trunk, such as marked variations in density and profile in the nerve fiber-associated dots or patches; it increased in intensity in the connective tissue covering the area of the motor endplate, as well as in the junctional myofiber surface. The structures recognizable by PNA coincided with components of the connective tissue such as collagen fibers and capillaries. Results suggest that: 1) the expression of PNA-binding molecules is dependent on the state of innervation, and 2) the spatiotemporal relationship between neural profiles and PNA staining provides sequences of axonal extension and subsequent nerve terminal maturation during regeneration in the motor endplate.

Morphological study of pancreatic endocrine in an experimental chronic pancreatitis with diabetes induced by stress and cerulein.

The purpose of this study was to investigate the morphological changes in the islets observed in a new chronic pancreatitis model with diabetes induced by repetition of cerulein injection plus water-immersion stress in rats. The rats of this model were treated with water-immersion stress for 5 h and two intraperitoneal injections of 20 micrograms/kg body weight of cerulein once a week for 16 weeks. In the stress and cerulein group, 62% of the islets exhibited infiltration of mononucleated cells, and/or peri- and intrainsular fibrosis. On immunohistochemical study, some islets showed reduced density of the insulin immunoreactivity. The glucagon-producing cells decreased in number. With electron microscopy, various endocrine changes were observed, mainly in the B cells. The changes included scattered debris damage with reduction of secretary granules, and vesiculation of the endoplasmic reticulum. Numerous fibroblasts clustered around the islets, and proliferating collagen fibers invaded the islets. The microvascular changes consisted of bleeding and damage to the endothels. In the pancreas treated with stress alone or cerulein alone, significant endocrine damage was not observed. In conclusion, chronic repetitive treatment with stress and cerulein, together with poor islet circulation due to fibrosis and vascular changes, resulted in the endocrine cellular damage.

Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes.

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.

Immunohistochemical distribution of c-Kit-positive cells and nitric oxide synthase-positive nerves in the guinea-pig small intestine.

Anatomical relationships between c-Kit-positive cells and nitric oxide synthase-positive nerves in the small intestine were examined by double-labeling immunohistochemistry. Cryosections and whole mount preparations of the guinea-pig small intestine were double-immunolabeled using anti-c-Kit and neuronal nitric oxide synthase antibodies, and were observed using confocal laser scanning microscopy. The c-Kit-like immunoreactivity constituted dense reticular networks in the deep muscular plexus and myenteric plexus of the intestinal wall. The nitric oxide synthase-like immunoreactivity occurred in the circular muscle layer, most densely at the deep muscular plexus, as well as within the ganglion strands or connecting strands of the myenteric plexus. Close association between c-Kit-like immunoreactivity and nitric oxide synthase-like immunoreactivity was evident in the deep muscular plexus. Specimens immunolabeled with the anti-nitric oxide synthase antibody were further examined under transmission electron microscopy. Axon profiles with nitric oxide synthase-like immunoreactivity lay closely adjacent to the interstitial cells in the deep muscular plexus as well as to smooth muscle cells of the circular muscle layer, whereas there was a considerable distance (> 500 nm) between interstitial cells and axon profiles with nitric oxide synthase-like immunoreactivity in the myenteric plexus. These results suggest that the interstitial cells in the deep muscular plexus serve as mediators of the nitrergic neurotransmission to the musculature in the small intestine, playing a role in the regulation of intestinal movement.

Effect of Ca2+-alginate gel dissolution on release of dextran with different molecular weights.

Release of macromolecular drug from calcium-alginate gel beads was investigated. Fluorescein isothiocyanate-labeled dextrans with molecular weight ranging from 9400 to 145000 were used as a model macromolecular drug. Dextran release was observed to be molecular weight-dependent, with the release pattern changing from pseudo first-order for dextran with 9400 molecular weight to sigmoidal for dextran with molecular weight of 145000. Release of a lower molecular weight dextran is mainly governed by the drug diffusion through the calcium-alginate gel matrix. With increasing dextran molecular weights, dextran release was strongly influenced by the dissolution of alginate matrix through the exchange of Ca2+ ions which act as a cross-linker. Rapid and complete release was achieved for dextran with 145000 molecular weight in a "burst" fashion showing a initial lag time. Minimal dextran release was observed in pseudo-gastric fluid at pH 1.2, while rapid dextran release within a narrow time range was achieved in simulated intestinal fluid at pH 6. 8. These results strongly suggest that calcium-alginate gel is a useful vehicle for pulsatile release of macromolecular drugs in oral drug delivery.

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon.

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement.